Oral Presentation 33rd ASM of the Australian & New Zealand Bone & Mineral Society 2023

Lysosomal proteins Legumain and Cathepsin B expressed within osteocytes cleave Collagen Type I (#7)

Natalie YY Koh 1 2 , Martha A. Blank 1 2 , Ingrid J. Poulton 2 , Alexander Ziegler 1 3 , Christopher S. Kovacs 4 , Natalie KY Wee 1 2 , Laura E. Edgington-Mitchell 1 3 , Natalie A. Sims 1 2
  1. University of Melbourne, Melbourne, VIC, Australia
  2. St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
  3. Bio21 Molecular Science and Biotechnology Institute, Parkville, Vic, Australia
  4. Faculty of Medicine-Endocrinology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada

During lactation (breast-feeding), to meet the neonate’s nutritional demands the maternal calcium is provided from the skeleton through bone resorption by osteoclasts on the bone surface. It has been suggested that osteocytes are also capable of removing their surrounding bone. This osteocytic osteolysis is activated by parathyroid hormone (PTH) and/or parathyroid hormone related-protein (PTHrP) signalling through PTH receptor 1 (PTHR1) on osteocytes. We sought to determine whether osteocytes, like osteoclasts, contribute to lactation-induced bone loss via lysosomal enzymes.

To identify osteocytic lysosomal enzymes, microarray datasets obtained from two previously published studies ((1) Kusa4b10s (osteoblasts) treated with PTH and PTHrP and (2) bones from age-matched lactating and unmated mice), were checked against 151 known lysosomal genes. Two were strongly upregulated by PTH and PTHrP: legumain and cathepsin B, the latter of which was also upregulated in lactating samples. 

Legumain and cathepsin B were detected by immunohistochemistry in tibial samples from (1) 6-week-old male C57BL/6 mice injected with 30 µg/kg hPTH(1-34) or vehicle and (2) lactating 10-week-old Black Swiss mice and age-matched controls. Both enzymes were detected in newly-embedded osteocytes in both sets of samples. Cathepsin B staining showed greater intensity in lactating bones compared to non-mated mice, and there was a 3-fold increase in the number of positively-stained osteocytes with PTH treatment compared to vehicle. 

To assess collagenolytic activity, recombinant legumain and cathepsin B were added to pure rat-tail collagen I and incubated at 37°C. Cleaved products were separated by gel electrophoresis and visualised by Coomassie stain. Both enzymes dose-dependently cleaved collagen I within 15 minutes. 

These data identify legumain and cathepsin B as collagen cleaving lysosomal enzymes that are expressed in osteocytes under normal physiological conditions and upregulated by PTH/PTHrP treatment and lactation. We conclude that osteocytes may use these lysosomal enzymes to conduct osteocytic osteolysis.